Abstract
Purpose: Few studies have investigated the properties of human orbital fibroblasts and no study has investigated whether these properties could provide insight into the pathogenesis of orbital inflammatory disease. Our purpose was to develop and refine a protocol for growing human orbital fibroblast cultures in vitro.
Method: A literature search was conducted for studies describing human fibroblast culture techniques. A draft protocol was tested and refined over a period of 12 months using human orbital fat specimens as the source of human orbital fibroblasts.
Results: Our final protocol for culturing human orbital fibroblasts is as follows:
Petri dishes are pretreated with 10 μg/mL poly‐D‐Lysine for 15 minutes at 37°C.
Retroseptal orbital fat specimens are collected from consenting patients undergoing orbital surgery at our institution and are transported to the laboratory fresh. Specimens are rinsed in phosphate buffered saline that has been autoclaved and filtered. Under sterile conditions inside a laminar flow cabinet, orbital specimens are dissected into 2 mm × 2 mm fragments, placed on the petri dishes, pressed flat with glass cover slips, and immersed in heat‐inactivated Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 50 μg/mL gentamicin sulfate.
Petri dishes are cultured at 37°C in 10% CO2 and the culture medium is changed every 7 days. Coverslips and fat fragments are removed at three weeks. Cells are passaged into flasks when they reach confluence, which occurs at approximately 6 weeks.
Using this protocol we have successfully established a large number of human orbital fibroblast cultures. The fibroblast phenotype has been confirmed by the observation of spindle‐shaped cell morphology and the expression of Fibroblast Surface Protein.
Conclusions: Human orbital fibroblast cultures can be established and maintained in vitro. This may serve as a useful experimental model from which to investigate orbital fibroblast physiology and the pathogenesis of orbital inflammatory disease.
Method: A literature search was conducted for studies describing human fibroblast culture techniques. A draft protocol was tested and refined over a period of 12 months using human orbital fat specimens as the source of human orbital fibroblasts.
Results: Our final protocol for culturing human orbital fibroblasts is as follows:
Petri dishes are pretreated with 10 μg/mL poly‐D‐Lysine for 15 minutes at 37°C.
Retroseptal orbital fat specimens are collected from consenting patients undergoing orbital surgery at our institution and are transported to the laboratory fresh. Specimens are rinsed in phosphate buffered saline that has been autoclaved and filtered. Under sterile conditions inside a laminar flow cabinet, orbital specimens are dissected into 2 mm × 2 mm fragments, placed on the petri dishes, pressed flat with glass cover slips, and immersed in heat‐inactivated Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 50 μg/mL gentamicin sulfate.
Petri dishes are cultured at 37°C in 10% CO2 and the culture medium is changed every 7 days. Coverslips and fat fragments are removed at three weeks. Cells are passaged into flasks when they reach confluence, which occurs at approximately 6 weeks.
Using this protocol we have successfully established a large number of human orbital fibroblast cultures. The fibroblast phenotype has been confirmed by the observation of spindle‐shaped cell morphology and the expression of Fibroblast Surface Protein.
Conclusions: Human orbital fibroblast cultures can be established and maintained in vitro. This may serve as a useful experimental model from which to investigate orbital fibroblast physiology and the pathogenesis of orbital inflammatory disease.
| Original language | English |
|---|---|
| Pages (from-to) | 86-86 |
| Number of pages | 1 |
| Journal | Clinical and Experimental Ophthalmology |
| Volume | 41 |
| Issue number | S1 |
| DOIs | |
| Publication status | Published - Nov 2013 |
| Externally published | Yes |
